Bioprinted meniscus implant and methods of using same

ABSTRACT

Provided herein are meniscus implant compositions, as well as method for making and using the same. The subject meniscus implants find use in repairing and/or replacing damaged or diseased meniscal tissue in a mammalian subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of the filing date of U.S.Provisional Patent Application Ser. No. 62/351,222, filed on Jun. 16,2016, the disclosure of which application is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The invention provides synthetic tissue structures and methods for theirfabrication and use, including artificial meniscus implants, comprisingprecisely patterned layers containing a variable synthetic tissue fiberstructure dispensed from a bioprinter.

BACKGROUND OF THE INVENTION

The meniscus is one of the most commonly damaged areas of the kneejoint, with a mean incidence of injury in the United States of 66injuries per 100,000 people. Complete or partial removal of the meniscusrelieves acute pain, but without adequate replacement, meniscus removalcan lead to damage of the articular cartilage of the knee, leading toosteoarthritis (OA). The meniscus typically demonstrates poor healingpotential, and none of the currently available meniscal replacementoptions meets the necessary load-bearing and biomechanical requirementsof this unique tissue, while also successfully engrafting into thesurrounding tissue to provide a long-term solution to meniscus injury.

The tissue engineering art has long sought to fabricate viable syntheticstructures capable of mimicking and/or replacing living organs andtissues using myriad materials and methods. Historically, cells andother biological materials were seeded into pre-formed three-dimensionalscaffolds imparting a desired structure, with the scaffold preferablybeing biodegradable or otherwise removable. See, e.g. U.S. Pat. No.6,773,713. Despite decades of development, however, significantchallenges remain with this approach in respect of effective cellseeding and growth, and the technique does not work for more complexphysiological structures involving more complicated spatial arrangementsof different cell types.

More recently, 3D printing, a form of additive manufacturing (AM), hasbeen applied to create three-dimensional objects directly from digitalfiles, wherein the object is built up layer-by-layer to achieve thedesired three dimensional structure. Initial efforts to adapt 3Dprinting techniques to the creation of cellular constructs and tissues,termed 3D bioprinting, also focused on initial printing of scaffoldmaterials independent of the direct seeding or subsequent printing ofthe cellular materials, consistent with the above convention. See, e.g.U.S. Pat. Nos. 6,139,574; 7,051,654; 8,691,274; 9,005,972 and 9,301,925.Unfortunately, however, the polymers typically employed to form theprior art scaffolds, while generally considered biocompatible, are notphysiologically compatible. As such, cell viability is sacrificed withthis approach in favor of the mechanical stability of the requisitescaffold.

In the meniscus implant art in particular, for example, Bakarich et al.described a system in which a combination of an alginate/acrylamide gelprecursor solution and an expoxy based UV-curable adhesive were combinedto form a printable matrix material. ACS Appl. Mater. Interfaces6:15998-16006 (2014). The printable matrix material was used in a 3Dbioprinting process to deposit a 2D layer of the matrix material alone,after which UV light was passed over the layer for one to five minutesto solidify it before depositing another layer on top. Due to thenon-physiologic nature of the acrylamide gel and epoxy-based UV-curablematrix components, however, living cells cannot be maintained in thismatrix material during the bioprinting process, and the resultingscaffold is still non-conducive to cell growth, differentiation andcommunication.

Alternative 3D bioprinting techniques have also been describedemphasizing the converse, wherein mechanical structure and printingfidelity are sacrificed in favor of cell viability. These bioprintingsystems create synthetic tissues by depositing cellular materials withina biocompatible matrix, which is then cross-linked or otherwisesolidified after deposition to create a solid or semi-solid tissuestructure. See, e.g., U.S. Pat. Nos. 9,227,339; 9,149,952; 8,931,880 and9,315,043; U.S. Patent Publication No. 2012/0089238; No. 2013/0345794;No. 2013/0164339 and No. 2014/0287960. With all of these systems,however, the temporal delay between the deposition and crosslinkingsteps invariably leads to a lack of control over the geometry of theprinted structure, as well as the cellular and matrix composition of thestructure. Moreover, cellular viability is often still compromised inany event by the subsequent cross-linking or solidification event.

As but one example of this problem, Markstedt et al. described a systemin which hydrogels, such as collagen, hyaluronic acid, chitosan andalginate were used in combination with non-physiologic reinforcing fibermaterials, such as nanofibrillated cellulose, as a bio-ink for 3Dbioprinting. BioMacromolecules 16:1489-96 (2015). This bio-ink isdeposited as a 2D layer of material, which is submerged in a divalentcation bath (CaCl₂)) to crosslink for ten minutes and solidify the firstlayer before depositing another layer on top. Although living cells weresuccessfully incorporated into their bio-ink, a cell viability analysisdemonstrated that the cell viability decreased significantly as a resultof the cross-linking process, from ˜95.3% before embedding, to ˜69.9%after embedding and crosslinking. Furthermore, a comparison tonon-printed controls revealed that the decrease in cell viability waslikely due to the preparation and mixing of the bio-ink itself, ratherthan the actual 3D printing process.

Accordingly, existing 3D bioprinting techniques and materials havefailed to satisfactorily resolve the technical conflict betweenstructural integrity and printing fidelity on the one hand, andphysiological compatibility and cellular viability on the other. Thecurrent invention addresses these and other unmet needs. All prior artreferences listed herein are incorporated by reference in theirentirety.

SUMMARY OF INVENTION

The present invention successfully resolves the previously conflictingobjectives in the 3D bioprinting art between structural integrity andcellular viability, providing synthetic tissue structures deposited insolidified form with improved cell growth and/or survivalcharacteristics and physiological functionality, and without the needfor cross-linking or other subsequent solidification steps. Aspects ofthe present invention include synthetic tissue structures comprising oneor more layers deposited by a bioprinter, wherein each layer comprisessynthetic tissue fiber(s) comprising a solidified biocompatible matrixoptionally comprising cells, and optionally comprising one or moreactive agents, wherein at least one of the matrix material, cell type,cell density, and/or amount of an active agent varies in at least onedirection within the layers. Preferably, at least one of said layerscomprises a single continuous synthetic tissue fiber dispensed from thebioprinter having a variable composition.

In specific embodiments, meniscus implants are provided comprisinglayers of synthetic tissue fiber(s) dispensed from a bioprinter as asolidified biocompatible matrix optionally comprising cells, andoptionally comprising one or more active agents, wherein at least one ofthe matrix material, cell type, cell density, and/or amount of an activeagent varies in at least one direction within the layers. Preferably, atleast one of said layers comprises a continuous synthetic tissue fiberdispensed from the bioprinter having a variable composition. Morepreferably, each of said layers comprises a continuous synthetic tissuefiber having a variable composition. Still more preferably, a meniscusimplant comprises a reinforced peripheral region, and/or at least oneanchor region, as described herein.

In one aspect, the invention provides a synthetic tissue structurecomprising a plurality of layers deposited by a bioprinter, each layercomprising synthetic tissue fiber(s) comprising a solidifiedbiocompatible matrix optionally comprising cells, and optionallycomprising one or more active agents, wherein at least one of saidlayers comprises a matrix material varying in type and/or amount in atleast one direction. In some embodiments, each layer comprises a matrixmaterial varying in type and/or amount in at least one direction.

In another aspect, the invention provides a synthetic tissue structurecomprising a plurality of layers, each layer comprising synthetic tissuefiber(s) comprising a plurality of mammalian cells dispensed from abioprinter within a solidified biocompatible matrix, wherein at leastone of said layers comprises a cell type and/or cell density varying inat least one direction. In some embodiments, each layer comprises a celltype and/or cell density varying in at least one direction.

In another aspect, the invention provides a synthetic tissue structurecomprising a plurality of layers deposited by a bioprinter, each layercomprising synthetic tissue fiber(s) comprising a solidifiedbiocompatible matrix optionally comprising cells, wherein at least oneof said layers comprises an active agent varying in type and/or amountin at least one direction. In some embodiments, each layer comprises anactive agent varying in type and/or amount in at least one direction.

In some embodiments, one or more synthetic tissue fibers are dispensedin a desired pattern or configuration to form a first layer, and one ormore additional layers are then dispensed on top, having the same or adifferent pattern or configuration. In certain embodiments, a pluralityof layers are stacked to form a three dimensional structure that can beused as an artificial meniscus implant. Preferably, at least one of saidlayers comprises a single continuous synthetic tissue fiber dispensedfrom the bioprinter having a variable composition. More preferably, eachof said layers comprises a single continuous synthetic tissue fiberhaving a variable composition.

In some embodiments, a synthetic tissue structure comprises a number ofindividual layers that ranges from about 1 to about 250, such as about2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,185, 190, 200, 205, 210, 215, 220, 225, 230, 235, 240 or about 245individual layers. Any suitable number of individual layers can beincorporated to generate a tissue structure having desired dimensions.

In some embodiments, one or more individual fibers and/or layers areorganized to create one or more zones within a tissue structure, whereineach zone has one or more desired properties (e.g., one or moremechanical and/or biological properties). As used herein, the term“region” refers to a portion of a tissue structure defined in an x-yplane (e.g., an area or portion of an individual layer, where each layerof the tissue structure defines an x-y plane), whereas the term “zone”refers to a portion of a tissue structure defined in the z-direction andcomprising at least two contiguous regions from separate x-y planes, orlayers (e.g., a “macrolayer” that comprises a plurality of individual“microlayers”).

Zones in accordance with embodiments of the invention can have anydesired three dimensional geometry, and can occupy any desired portionof a synthetic tissue structure. For example, in some embodiments, azone can span an entire length, width, or height of a synthetic tissuestructure. In some embodiments, a zone spans only a portion of a length,width, or height of a synthetic tissue structure. In some embodiments, asynthetic tissue structure comprises a plurality of different zones thatare organized along a length, width, height, or a combination thereof,of the synthetic tissue structure. In one preferred embodiment, asynthetic tissue structure comprises three different zones that areorganized along the height of the synthetic tissue structure, such thata path through the synthetic tissue structure from the bottom to the topwould pass through all three zones.

In some embodiments, a zone can comprise a number of layers that rangesfrom about 2 to about 250, such as about 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 205, 210, 215,220, 225, 230, 235, 240 or about 245 individual layers. In someembodiments, the individual layers within a zone are organized in amanner that confers one or more mechanical and/or biological propertieson the zone. For example, in some embodiments, the individual layerswithin a zone comprise one or more reinforcing materials that conferincreased mechanical strength on the zone. In some embodiments, theindividual layers within a zone comprise one or more materials thatconfer desirable cell growth properties on the zone. In someembodiments, the individual layers within a zone, or the plurality ofindividual compartments of a fiber structure passing through the zone,can be alternated in a manner that confers desirable properties on thezone. For example, in some embodiments, the individual layers or regionswithin a zone are alternated such that the odd numbered layers containone or more reinforcing materials that confer desirable mechanicalproperties on the zone, and the even numbered layers contain one or morematerials that confer desirable biological properties on the zone (e.g.,softer materials that are conducive to cell migration, growth,viability, and the like). In some embodiments, a zone comprises aplurality of contiguous individual layers (e.g., about 2, 3, 4, 5, 6, 7,8, 9 or about 10 or more contiguous layers) that comprise one or morereinforcing materials that confer increased mechanical strength on thezone, which contiguous layers are alternated with another plurality ofcontiguous individual layers (e.g., about 2, 3, 4, 5, 6, 7, 8, 9 orabout 10 or more contiguous layers) that comprise one or more materialsthat confer desirable biological properties on the zone (e.g., softermaterials that are conducive to cell migration, growth, viability, andthe like).

In one aspect, an artificial meniscus implant comprises at least onebasal zone, at least one interior zone, and at least one superficialzone, wherein at least one of said zones comprises a layer comprising asynthetic tissue fiber(s) comprising a solidified biocompatible matrix,wherein the matrix materials vary in type and/or amount between thecenter of a layer and the periphery of the layer. In some embodiments,one or more matrix materials at or near the periphery of the layercomprise a reinforced matrix material.

Aspects of the invention also include artificial meniscus implants thatcomprise one or more anchor regions. As used herein, the term “anchorregion” refers to a region that comprises one or more reinforced matrixmaterials. Artificial meniscus implants in accordance with embodimentsof the invention can include any suitable number of anchor regions, suchas 1 to 12, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 anchor regions. Insome embodiments, an artificial meniscus implant comprises no anchorregions.

In another aspect, artificial meniscus implants are provided comprisingat least one basal zone, at least one interior zone, and at least onesuperficial zone, wherein at least one zone comprises a layer comprisingat least one synthetic tissue fiber comprising a plurality of mammaliancells dispensed from a bioprinter within a solidified biocompatiblematrix, wherein at least one layer comprises a cell density that variesin at least one direction. In some embodiments, each of said layerscomprises a cell density that varies in at least one direction. In someembodiments, the cell density ranges from 0 to about 100×10⁶ cells/mL.

In another aspect, artificial meniscus implants in accordance withembodiments of the invention include at least one basal zone, at leastone interior zone, and at least one superficial zone, wherein at leastone layer in one of said zones comprises a synthetic tissue fiber(s)comprising a solidified biocompatible matrix and at least one activeagent, wherein the at least one active agent varies in type and/oramount between the center of the layer and the periphery of the layer.

In some embodiments, the biocompatible matrix on the periphery of thelayer may comprise at least one active soluble agent that is releasedover time from the matrix to encourage host vascular cell ingrowth andchondrocyte cell ingrowth. Such bioactive agents include, but are notlimited to: vascular endothelial growth factor (VEGF), fibroblast growthfactor (FGF), insulin-like growth factor-1 (IGF-1), bone morphogeneticfactors, hepatocyte scatter factor, urokinase plaminogen activator,transforming growth factor-β (TGF-β), platelet derived growth factor(PDGF), or any combination thereof.

In some embodiments, the biocompatible matrix on the periphery of thelayer may comprise at least one insoluble factor to encourage cellingrowth. Non-limiting examples of such insoluble factors include:hyaluronic acid or sulfated hyaluronic acid, fibronectin, fibrin, andcollagen I. Additional bioactive factors can be incorporated into thematrix arranged in the interior of the subject artificial meniscusimplants to encourage collagen deposition by chondrocytes including.Non-limiting examples of such additional bioactive factors include:insulin, connective tissue-derived growth factor (CTGF), or acombination thereof.

In some embodiments, portions or regions of the periphery will compriseat least one active agent. In some embodiments, the entire periphery ofa layer comprises at least one active agent. In some embodiments, theperiphery comprises a plurality of active agents. In some embodimentsthe entire periphery of the layer includes an active agent that reducesthe host inflammatory response, for example, via the inclusion of one ormore steroid compounds contained within one of more microparticles toensure sustained release over an extended time period.

In some embodiments, an artificial meniscus implant has an arcuate shapethat has an anterior end, a posterior end, a middle section therebetweendefining a curved path between said anterior and posterior ends, aninternal side, and an external side. In some embodiments, the celldensity increases in a radial manner from the internal side towards theexternal side. In some embodiments, the concentration of reinforcedmatrix materials increases in a radial manner from the internal sidetowards the external side. In some embodiments, the amount of activeagent increases in a radial manner from the internal side towards theexternal side.

In some embodiments, the basal zone comprises one or more layerscomprising randomly-oriented synthetic tissue fiber(s); the interiorzone comprises one or more layers comprising circumferentially-orientedsynthetic tissue fiber(s) and radially-oriented synthetic tissuefiber(s); and the superficial zone comprises one or more layerscomprising randomly-oriented synthetic tissue fiber(s). In someembodiments, the circumferentially-oriented synthetic tissue fiber(s)has a first diameter and the radially-oriented synthetic tissue fiber(s)has a second, different diameter. In some embodiments, thecircumferentially-oriented synthetic tissue fiber(s) and theradially-oriented synthetic tissue fiber(s) have the same diameter. Insome embodiments, the synthetic tissue fiber(s) has a diameter thatranges from about 20 μm to about 500 μm.

In some embodiments, the circumferentially-oriented synthetic tissuefiber(s) comprises a first solidified biocompatible matrix, and theradially-oriented synthetic tissue fiber(s) comprises a second,different solidified biocompatible matrix. In some embodiments, thecircumferentially-oriented synthetic tissue fiber(s) and theradially-oriented synthetic tissue fiber(s) comprise the same solidifiedbiocompatible matrix.

In some embodiments, the interior zone comprises a layer comprising asynthetic tissue fiber(s) that is configured to promote deposition ofcollagen fibers aligned with a longitudinal direction of the synthetictissue fiber(s). In some embodiments, the interior zone comprises alayer comprising a circumferentially-oriented synthetic tissue fiber(s)that is configured to promote deposition of collagen fibers that arealigned with a longitudinal direction of the circumferentially-orientedsynthetic tissue fiber(s). In some embodiments, the interior zonecomprises a layer comprising a radially-oriented synthetic tissuefiber(s) that is configured to promote deposition of collagen fibersthat are aligned with a longitudinal direction of the radially-orientedsynthetic tissue fiber(s).

The solidified biocompatible matrix can comprise any of a wide varietyof natural or synthetic polymers that support the viability of livingcells, including, e.g., alginate, laminin, fibrin, hyaluronic acid,poly(ethylene) glycol based gels, gelatin, chitosan, agarose, orcombinations thereof. In preferred embodiments, the solidifiedbiocompatible matrix comprises alginate, or other suitable biocompatiblepolymers that can be instantaneously solidified while dispensing fromthe print head. In further preferred embodiments, the solidifiedbiocompatible matrix comprises a homogeneous composition of alginatethroughout the radial cross section of each synthetic tissue fiber.

In particularly preferred embodiments, the solidified biocompatiblematrix is physiologically compatible, i.e., conducive to cell growth,differentiation and communication. In some such embodiments, thephysiologically compatible matrix comprises alginate in combination withone or more of: collagen, fibronectin, thrombospondin,glycosaminoglycans (GAG), deoxyribonucleic acid (DNA), adhesionglycoproteins, elastin, and combinations thereof. In specificembodiments, the collagen is selected from the group consisting of:collagen I, collagen II, collagen III, collagen IV, collagen V, collagenVI, or collagen XVIII. In specific embodiments, the GAG is selected fromthe group consisting of: hyaluronic acid, chondroitin-6-sulfate,dermatan sulfate, chondroitin-4-sulfate, or keratin sulfate.

As reviewed above, anchor regions can be generated by the incorporationof higher strength materials into specific zones of an implant (i.e.,suture points), for example, stiffer synthetic materials, including, butnot limited to, polycaprolactone (PCL), poly(lactic-co-glycolic acid)(PLGA), polyurethane (PU) and any combination thereof. In someembodiments, an anchor region can contain double network hydrogels,generated by combining at least two different hydrogel materials,examples of which include, without limitation, alginate, Gelatinmethacrylol (GelMA), methacryloyl polyethylene glycol (PEGMA), gellangum, agarose, polyacrylamide, or any combination thereof. In addition,high strength fibers can be generated from high concentrations ofbiological polymers including, without limitation, collagen, chitosan,silk fibroin, or any combination thereof, and these biological polymerscan be incorporated into one or more anchor regions. In someembodiments, an anchor region and/or a reinforced peripheral region ofan implant comprises one or more layers of high strength material(s)deposited in alternation in the z-direction with one or more layers ofsofter matrix materials containing, e.g., hydrogel material(s) conduciveto cell survival and ingrowth described above. In this way, the softer,cell compatible hydrogel materials provide one or more desirablebiological functions, and the stiffer materials provide one or moredesirable mechanical functions, to generate a hybrid structure withappropriate mechanical and biological functions.

In some embodiments, the mammalian cells are selected from the groupconsisting of: fibroblasts, chondrocytes, fibrochondrocytes, primaryhuman meniscus-derived chondrocytes, stem cells, bone marrow cells,embryonic stem cells, mesenchymal stem cells, bone marrow-derivedmesenchymal stem cells, induced pluripotent stem cells, differentiatedstem cells, tissue-derived cells, microvascular endothelial cells, andcombinations thereof. In preferred embodiments, the cell viabilitywithin the synthetic living tissue structures ranges from about 70% upto about 100%, such as about 75%, about 80%, about 85%, about 90%, about95%, about 98%, about 99%, about 99.5%, or about 99.9% in comparisonwith cell viability before printing.

In some embodiments, the meniscus implant further comprises an acellularsheath positioned below the basal zone. In some embodiments, themeniscus implant further comprises an acellular sheath positioned abovethe superficial zone. In some embodiments, the meniscus implantcomprises a first acellular sheath positioned below the basal zone and asecond acellular sheath positioned about the superficial zone.

In some embodiments, the meniscus implant further comprises at least oneactive agent. In some embodiments, the at least one active agent isselected from the group consisting of: TGF-β1, TGF-β2, TGF-β3, BMP-2,BMP-4, BMP-6, BMP-12, BMP-13, basic fibroblast growth factor, fibroblastgrowth factor-1, fibroblast growth factor-2, platelet-derived growthfactor-AA, platelet-derived growth factor-BB, platelet rich plasma,IGF-I, IGF-II, GDF-5, GDF-6, GDF-8, GDF-10, vascular endothelialcell-derived growth factor, pleiotrophin, endothelin, nicotinamide,glucagon like peptide-I, glucagon like peptide-II, parathyroid hormone,tenascin-C, tropoelastin, thrombin-derived peptides, laminin, biologicalpeptides containing cell-binding domains and biological peptidescontaining heparin-binding domains, therapeutic agents, and anycombinations thereof.

In preferred embodiments, the bioprinter dispenses the solidifiedbiocompatible matrix comprising the plurality of mammalian cells througha single orifice. In particularly preferred embodiments, the singleorifice is comprised within a print head such as that described andclaimed in WO 2014/197999, the disclosure of which is hereinincorporated by reference in its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of a layer-by-layer synthetic tissuefiber deposition process.

FIG. 2 is a schematic illustration of a knee joint, depicting a lateraland a medial meniscus. (Adapted from: The knee meniscus:structure-function, pathophysiology, current repair techniques, andprospects for regeneration. Biomaterials. 2011 October; 32(30):7411-7431. doi:10.1016/j.biomaterials.2011.06.037, Eleftherios A.Makris, MD1, Pasha Hadidi, BS1, and Kyriacos A. Athanasiou, Ph.D.,P.E.1).

FIG. 3 is a schematic illustration of a meniscus, depicting both a topview and a cross sectional view. The outer (red-red) region, central(white-red) region, and the inner (white-white) region are depicted.(Adapted from: The knee meniscus: structure-function, pathophysiology,current repair techniques, and prospects for regeneration. Biomaterials.2011 October; 32(30): 7411-7431. doi:10.1016/j.biomaterials.2011.06.037,Eleftherios A. Makris, MD1, Pasha Hadidi, BS1, and Kyriacos A.Athanasiou, Ph.D., P.E.1).

FIG. 4 , Panel A is a schematic illustration of a meniscus, depicting across sectional view. Circumferential and radial alignment of collagenfibers confer biomechanical properties to the meniscus. Panel B depictsa superficial zone, a lamellar zone, and an interior (deep) zone.Collagen fibers in the superficial and lamellar zones close to themeniscus surface are randomly oriented. Fibers deeper in the meniscusare oriented in both circumferential and radial directions.

FIG. 5 is a force diagram that depicts the components of an axial loadforce F on various portions of a meniscus. The axial load force (F)perpendicular to the meniscus surface and horizontal force (fr) arecreated by compressing the femur (F_(f)). F rebounds due to the tibialupgrade force (Ft), whereas fr leads to meniscal extrusion radially,which is countered by the pulling force from the anterior and posteriorinsertional ligaments. Consequently, tensile hoop stress is createdalong the circumferential directions during axial compression, which isresisted by the circumferentially-oriented collagen fibers. (Adaptedfrom: The knee meniscus: structure-function, pathophysiology, currentrepair techniques, and prospects for regeneration. Biomaterials. 2011October; 32(30): 7411-7431. doi:10.1016/j.biomaterials.2011.06.037,Eleftherios A. Makris, MD1, Pasha Hadidi, BS1, and Kyriacos A.Athanasiou, Ph.D., P.E.1).

FIG. 6 provides images of two cell-free 3D meniscus-like structures withpre-programmed zone-specific scaffold content and coordinated patterningof printed synthetic tissue fiber structures. Scale bar=1 cm.

FIG. 7 is a series of microscope images that depict spontaneous collagenfiber alignment in small diameter fibers. Polymerised collagen fiberorientation in microfluidic channels of different diameters including;30 um (Panel a), 100 um (Panel b), 400 um (Panel c) and no channel(Panel d) (Lee et al., 2006).

FIG. 8 shows a series of images showing on-the-fly modulation of printedalginate-based fiber diameter using a 3D bioprinting system, as well asa graph comparing the mean fiber diameters. Panels A, B, and C depictalginate-based fibers of 3 different diameters that were generated byprinting at 3 different pressure settings in the 3D bioprinting system.Quantification of width in multiple fibers demonstrates that the meandiameter at each pressure setting is consistent (graph, right).

FIG. 9 is an illustration of synthetic tissue fiber patterning invarious layers of a 3D bioprinted meniscus. Synthetic tissue fiberstructures of specific diameters loaded with extracellular matrix (ECM),e.g., collagen, are patterned in a manner that recapitulates themicro-patterning of collagen and the zonal architecture of the meniscus.The basal and superficial zones contain randomly oriented fibers printedin larger diameter fibers. The interior zones contain circumferentialand radially-aligned collagens aligned within patterned fibers ofsmaller diameter.

FIG. 10 shows data from 2-photon imaging of collagen fibers in anengineered 3D tissue. Panel A: formaldehyde-fixed, H&E stained sectionof a 3D co-culture of primary human airway epithelial cells andfibroblasts after 90 day culture on an electrospun gelatin (ESG)scaffold. Panel B: 2-photon imaging of unstained sections demonstratingdeposition of fibrillar collagen (purple) oriented parallel to thesurface of the ESG scaffold, in a similar direction to the fibroblastsdepositing the collagen. Panel C: Emission spectra of the unstainedtissues demonstrates that non-centro-symmetric collagen fibers generatea specific 2^(nd) harmonic signal (SHG) (Wadsworth et al., 2014).

FIG. 11 is an illustration of a meniscal tissue with zone-specific celland ECM content. “Red-Red bio-ink” and “White-white bio-ink” are used togenerate tissues with zonal architecture. Desired cell types, (e.g.,MSC-derived chondrocytes or primary meniscus-derived cells) are seededat appropriate physiological densities into red-red and white-whitezones. Specific ECM content of the scaffold is modified according to thetissue zone. The “white-red” zone in the central zone of the tissuecontains a mixture of red-red and white-white bio-inks and cells. Thebioprinting system facilitates control over both the cellular (cell typeand cell density) and ECM content in any given zone of the meniscusimplant.

DETAILED DESCRIPTION

Aspects of the present invention include synthetic tissue structurescomprising one or more layers deposited by a bioprinter, wherein eachlayer comprises synthetic tissue fiber(s) comprising a solidifiedbiocompatible matrix optionally comprising cells, and optionallycomprising one or more active agents, wherein at least one of the matrixmaterial, cell type, cell density, and/or amount of an active agentvaries in at least one direction within the layers. Preferably, at leastone of said layers comprises a single continuous synthetic tissue fiberdispensed from the bioprinter having a variable composition. The term“solidified” as used herein refers to a solid or semi-solid state ofmaterial that maintains its shape fidelity and structural integrity upondeposition. The term “shape fidelity” as used herein means the abilityof a material to maintain its three dimensional shape. In someembodiments, a solidified material is one having the ability to maintainits three dimensional shape for a period of time of about 30 seconds ormore, such as about 1, 10 or 30 minutes or more, such as about 1, 10,24, or 48 hours or more. The term “structural integrity” as used hereinmeans the ability of a material to hold together under a load, includingits own weight, while resisting breakage or bending.

In some embodiments, a solidified composition is one having an elasticmodulus greater than about 15, 20 or 25 kilopascals (kPa), morepreferably greater than about 30, 40, 50, 60, 70, 80 or 90 kPa, stillmore preferably greater than about 100, 110, 120 or 130 kPa. Preferredelastic modulus ranges include from about 15, 25 or 50 Pa to about 80,100, 120 or 140 kPa.

Additional aspects of the invention include artificial meniscus implantsfor use in repairing and/or replacing a damaged or diseased meniscaltissue in a mammalian subject, comprising synthetic tissue fiber(s)dispensed from a bioprinter as a solidified biocompatible matrixoptionally containing cells, and optionally containing one or moreactive agents, wherein at least one of the matrix material, cell type,cell density, and/or type and/or amount of an active agent varies in atleast one direction within the fiber.

As provided in FIG. 1 , a solidified biocompatible matrix optionallycontaining a plurality of mammalian cells is dispensed from a bioprinterforming one or more synthetic tissue fiber(s) on a deposition surface,and ultimately forming a layer. As such, subsequent cross-linking orother solidification steps are unnecessary after dispensation of thealready-solidified matrix from the printhead. Accordingly, a secondlayer can be rapidly deposited on top of the first layer, whilemaintaining the structural integrity of the first layer, and thisprocess can be continued to deposit a plurality of layers, one on top ofthe next, until a three dimensional structure having a desired geometryis obtained.

The solidified biocompatible matrix may advantageously comprisealginate, or any other suitable biocompatible polymer that can beinstantaneously solidified while dispensing from the printhead. In apreferred embodiment, the alginate-based matrix is printed andsimultaneously crosslinked at the time of printing by contacting with adivalent cation crosslinking solution (e.g., a CaCl₂) solution) beforeor upon dispensation from the printhead. In particularly preferredembodiments, the alginate-based biocompatible matrix further comprisesone or more physiological materials, as described in more detail herein.In further preferred embodiments, the solidified biocompatible matrixcomprises a homogeneous composition of alginate throughout the radialcross section of each synthetic tissue fiber.

In some embodiments, a synthetic tissue fiber structure comprises aplurality of individual compartments (organized along the length of thesynthetic tissue fiber) that are created by sequentially depositingdifferent matrix materials (e.g., natural and/or synthetic polymers),different cell types, different cell concentrations, and/or differenttypes and/or amounts of active agents in each compartment of the samecontinuous synthetic tissue fiber structure. For example, in someembodiments, a synthetic tissue fiber structure comprises a firstcompartment that comprises a first matrix material, and a secondcompartment that comprises a second matrix material. In someembodiments, a synthetic tissue fiber structure comprises a firstcompartment that comprises a first cell type, and a second compartmentthat comprises a second cell type. In some embodiments, a synthetictissue fiber structure comprises a first compartment that comprises afirst cell concentration, and a second compartment that comprises asecond cell concentration. In some embodiments, a synthetic tissue fiberstructure comprises a first compartment that comprises a first activeagent, and a second compartment that comprises a second active agent.Any combination of matrix materials, cell types, cell concentrations,and/or types and/or amounts of active agents can be used in differentcompartment of a subject synthetic tissue fiber structure to achievedesired biomechanical properties and/or biological activities.

Synthetic tissue fiber structures in accordance with embodiments of theinvention can include controlled patterning of different matrixmaterials (e.g., natural and/or synthetic polymers) and crosslinkingtechniques to create a desired cross-sectional profile within a givencompartment. For example, in some embodiments, a synthetic tissue fiberstructure comprises a compartment having a solid, tubular, or porouscross-sectional profile. Non-limiting examples of cross-sectionalprofiles that can be created in a synthetic tissue fiber structure inaccordance with embodiments of the invention include those described inJun, Yesl, et al. “Microfluidic spinning of micro- and nano-scale fibersfor tissue engineering.” Lab on a Chip 14.13 (2014): 2145-2160, thedisclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the resulting synthetic tissue fiber is patterned,using software tools, to form layers optionally containing a pluralityof mammalian cells and/or a plurality of biocompatible matrix materials.In certain embodiments, a plurality of layers is deposited in asequential manner to generate a multi-layered meniscus implantcomprising a plurality of zones. In some embodiments, a meniscus implantcomprises at least one basal zone, at least one interior zone, and atleast one superficial zone, wherein the interior zone comprises at leastone layer comprising at least one circumferentially-oriented synthetictissue fiber, and at least one radially-oriented synthetic tissue fiber.Preferably, at least one of said layers comprises a single continuoussynthetic tissue fiber dispensed from the bioprinter having a variablecomposition.

One advantage of the subject meniscus implants is that the matrixcomposition, cell type, cell density, and active agent type and/orconcentration can be controlled at any given point in any portion of anylayer of the implant, thereby facilitating the generation of meniscusimplants more closely resembling the natural architecture of a meniscustissue, and that possess desirable biomechanical properties, including,but not limited to, reinforced anchor regions on the periphery of theimplant, circumferentially- and radially-oriented fiber structureswithin the meniscus implant, as well as specific cell types and celldensities within specific regions and/or zones of the implant.

Another advantage of the present invention is that one or more activeagents (described in more detail herein) can be selectively added todifferent compartments of a synthetic tissue fiber to allow preciselocalization of an active agent within one or more layers of a meniscusimplant, including, but not limited to, increased concentrations ofappropriate active agents on the periphery of an acellular implant toencourage the ingrowth of endogenous cells. The subject meniscusimplants are described in further detail below.

Meniscus Anatomy:

The menisci are a pair of crescent-shaped fibrocartilages comprised ofboth a medial and a lateral component situated between the correspondingfemoral condyle and tibial plateau. (FIG. 2 ). The anterior andposterior insertional ligaments attach the menisci firmly, and they fixthe meniscus to the tibial plateau well. Menisci are generallywedge-shaped, and the lateral menisci are approximately 32.4-35.7 mm inlength, and approximately 26.6-29.3 mm wide, while the medial menisciare approximately 40.5-45.5 mm long and approximately 27 mm wide. Eachis a glossy-white, complex tissue comprised of cells, specializedextracellular matrix (ECM) materials, and zone-specific innervation andvascularization. The menisci are fully vascularized at birth, however,over time the blood vessels retreat outwards until (in humans) at 10years of age, approximately 10-30% of the meniscus at the periphery isvascularized. The adult human meniscus thus has two distinct zones, theouter, vascular/neural zone (red-red zone), and the inner completelyavascular/aneural zone (white-white zone). These regions are separatedby the narrow central (red-white) zone that contains features of boththe outer (red-red) and the inner (white-white) zones (FIG. 3 ).Critically, the self-healing capacity of each area is directly relatedto blood supply, leaving the inner, white-white zone susceptible totrauma and degenerative lesions.

Meniscus Cellular and Biochemical Composition

The meniscus is a highly hydrated tissue comprising approximately 72%water, with the remaining 28% mostly comprising ECM and cells. Collagensmake up most of the ECM (75%) followed by glycosaminoglycans (GAGs, 17%)DNA (2%), adhesion glycoproteins (<1%) and elastin (<1%). These ratiosvary depending on the zone of the tissue, age, and condition. Thecellular component of the meniscus is zone-specific, comprising bothfibrochondrocytes and chondrocyte-like cells.

The composition of the meniscus differs in each zone. In the outerred-red zone, the cells are more fibroblast-like in morphology, withmany processes. The ECM in this zone is mainly fibrillar collagen type-I(80%). The inner white-white zone has ECM closely resembling hyalinecartilage, with more collagen-II (42%), a reduced proportion ofcollagen-I (28%) and a higher GAG concentration. The cells in this zoneare termed fibrochondrocytes, or chondrocyte-like cells. The superficiallayers of the menisci have another distinct cell type with potentialstem cell-like properties. The zone-specific ECM components of themeniscus are generated by the cells resident within the tissue, thusphenotypic markers for meniscal cells can include ECM protein expressionor gene expression such as: COL1A1 (collagen-1), COL2A2 (collagen-2),VCAN (versican), ACAN (aggrecan), CSPG4 (chondroitin-6-sulfate), Sox9and Col10a (collagen-10a). Similar to the unique cell types in eachmeniscal zone, cell density also varies in each zone. Vascular (red-red)and avascular (white-red, white-white) zones have average cell densitiesof 12,820 cells/mm³ and 27,199 cells/mm³, respectively, and morefibrochondrocytes than fibroblast-like cells (Cengiz et al., 2015). Themeniscus is highly heterogenous, with zone-specific variation in cellphenotype and ECM composition.

The heterogeneous distribution of cell types and biochemical scaffoldcontent of the knee meniscus is described in Table 1. The red-red zoneis characterised by fibroblast-like cells and a collagen-I-predominantextracellular matrix (ECM), with trace amounts of collagen-II. Thewhite-red and white-white zones contain fibrochondrocyte cells and amatrix rich in collagen-II, and a higher proportion ofglycosaminoglycans (GAGs).

TABLE 1 Zone Organic Red-Red component zone Red-white zone White-Whitezone Cells Vessels, Fibrochondrocytes/ Fibrochondrocytes nerves, &Chondrocyte-like & superficial zone fibroblast- cells cells (stem cells)like cells ECM (% total dry wgt) Total collagen    >80% 70% 70%Collagen-I    >80% 28% 28% Collagen-II     <1% 42% 42% Collagens-III,    <1% <1% <1% IV, V, VI, XVIII, fibronectin, thrombospondin Elastin <0.6% <0.6%  <0.6%  GAGs (% total dry wgt) Total GAGs    17% 30% 30%Chondroitin-6-   10.2% 18% 18% sulfate Dermatan sulfate 3.4-5.1%6.0-9.0%    6.0-9.0%    Chondroitin-4- 1.7-3.4% 3.0-6.0%    3.0-6.0%   sulfate Keratin sulfate     2.6% 4.5%  4.5% Collagen Fiber Patterning Confers Meniscal Biomechanical Properties

The micro-anatomic geometry of the meniscus is closely associated withits biomechanical properties. The hydrated nature of the meniscus (˜72%water) confers resistance to compressive stress, as water isincompressible, however, the meniscus has considerable tensile strengthwhich is conferred via the ordered arrangement of 10 μm-diametercollagen fibers throughout the tissue (FIG. 4 ) (Baker et al., 2007).The surface and lamellar zones of the meniscus are made up of randomlyoriented collagen fibers, whereas the fibers deeper in the meniscus areoriented in circumferential and radial directions. With normal use,forces of several times body weight arise within the knee, with themenisci transmitting 50-100% of this load through the dense network ofcircumferentially aligned collagen fibers (FIG. 4 ). This orderedarchitecture engenders very high tensile properties in the fiberdirection (50-300 MPa) (Baker et al., 2007). Tensile hoop stress iscreated in the circumferential direction when the knee bears an axialload, and this stress tries to extrude the meniscus out of the kneejoint (FIG. 5 ). However, the tensile strength ofcircumferentially-aligned collagen fibers and the firm attachment at theanterior and posterior insertional ligaments helps prevent extrusion ofthe meniscus and significantly reduces stress and protects the tibialcartilage. In contrast, if the anterior or posterior insertionalligaments or peripheral circumferential collagen fibers rupture, theload transmission mechanism changes, which damages the tibial cartilage.Compressive strength has been measured in fresh-frozen cadaveric humanmenisci, the axial and radial compressive moduli at 12% strain were 83.4kPa and 76.1 kPa, respectively, with tensile modulus several orders ofmagnitude greater (Chia & Hull, 2008).

The goal of tissue engineering is to generate a structure thatrecapitulates the function of the native tissue. In the case of themeniscus, the challenge is to generate a living tissue capable oflong-term engraftment into the knee joint, while also having thebiomechanical strength necessary to withstand the considerablecompressive forces that it is exposed to during everyday life. Themeniscus is a surprisingly complex tissue with specific architecture atthe mm, μm and nm scale, all of which contribute to the biomechanicalfunction of the tissue. To date, meniscal engineering has been somewhatlimited by the fabrication tools available to researchers, such asmolding hydrogels using casts, or seeding cells onto prefabricatedscaffolds. These approaches are not capable of generating themicro-scale architectures necessary to recapitulate function. Incontrast, the meniscus implants described herein are able to achievepoint to point control over matrix material(s), cell type, cell density,and active agent composition, which facilitates the generation of animplant that more closely resembles native structural features of themeniscus.

The meniscus is a heterogeneous tissue, with cells and ECM componentsdistributed in specific zones. Zonal specificity is vital for conferringregenerative and biomechanical function. The subject artificial meniscusimplants employ specific placement of different matrix materials, celltypes, cell densities, and active agent compositions into preciseregions and/or zones of the 3D tissue, thus allowing for re-creation ofthe red-red, white-red, white-white zonal architecture of the meniscus(FIG. 6 ).

The density of cells within the human meniscus has been demonstrated tovary in a zone-specific manner (approximately 13×10⁶ cells/ml in thered-red zone, and 28×10⁶ cells/ml in the white-white and white-red zones(Cengiz et al., 2015)). Cell density plays a vital role in maintainingappropriate cell phenotype, ECM organization and corresponding tissuebiomechanics. In some embodiments, the subject meniscus implantscomprise cell densities ranging from about 0 to about 100×10⁶ cells/mLor more. As such, in some embodiments, the subject meniscus implants canhave a cell density that varies from one position within the implant toanother. For example, in certain embodiments, a meniscus implantcomprises a layer having a cell density that varies in at least onedirection. In other embodiments, the subject implants are acellular anddesigned for endogenous cell ingrowth.

Collagen gives most tissues tensile strength, and multiple collagenfibrils approximately 100 nm in diameter combine to generate strongcoiled-coil fibers of approximately 10 μm in diameter. Biomechanicalfunction of the meniscus is conferred via collagen fiber alignment incircumferential and radial directions (FIG. 4 ). In some embodiments,the subject meniscus implants comprise patterned collagen fibrils thatare created by modulating the diameter of the synthetic tissue fiberstructures that are used to create the implant.

Previous studies have shown that microfluidic channels of differentdiameters can direct the polymerization of collagen fibrils to formfibers that are oriented along the length of the channels, but only atchannel diameters of 100 μm or less (Lee et al., 2006) (FIG. 7 ).Primary endothelial cells grown in these oriented matrices were shown toalign in the direction of the collagen fibers. In another study,Martinez et al, demonstrate that 500 um channels within a cellulose-beadscaffold can direct collagen and cell alignment (Martinez et al., 2012).In some embodiments, the subject meniscus implants comprise synthetictissue fiber structures that have a diameter that ranges from about 20μm to about 500 μm, such as about 50 μm, about 75 μm, about 100 μm,about 125 μm, about 150 μm, about 175 μm, about 200 μm, about 225 μm,about 250 μm, about 275 μm, about 300 μm, about 325 μm, about 350 μm,about 375 μm, about 400 μm, about 425 μm, about 450 μm, or about 475 μm(FIG. 7 ). By modulating the fiber diameter, the orientation of thecollagen fibers within the subject meniscus implants can be controlled.As such, the synthetic tissue fiber structures, and the collagen fiberswithin them, can therefore be patterned to produce meniscus implantswith a physiologically accurate arrangement of circumferential andradially aligned collagen fibers, essential for conferring necessarybiomechanical properties on the meniscus implants (FIG. 8 ).

The meniscus is an intrinsically heterogeneous structure with zones ofvarying composition and architecture. The subject meniscus implantscomprise complex biological structures that comprise unique materialcompositions and architectures, including, without limitation, fiberdiameter, ECM composition, cell composition, and cell density. Theability to control these and other aspects of the synthetic tissue fiberstructures that are used to generate the subject meniscus implantsenables construction of the zonal architectures found in native meniscustissue.

In certain embodiments, the subject meniscus implants are generatedusing automated control systems that modulate one or morecharacteristics of the synthetic tissue fiber(s) to achieve, e.g.,material switching within an individual fiber structure, betweenseparate fiber structures, within or across a layer, within or across azone, and essentially at any point throughout the structure. As aresult, point to point control of the meniscus implant composition isachieved. Furthermore, key parameters, such as fiber diameter and layerthickness, can also be modulated as desired. This level of automatedcontrol is essential to accurately recreating the heterogeneouscomposition and morphology found in native knee menisci.

The subject synthetic tissue fibers support the viable growth of a widevariety of human cells. The synthetic tissue fiber structures can befinely tuned to contain, e.g., different ECM proteins, GAGs and growthfactors to optimize the matrix for specific cell types.Computer-controlled deposition of the synthetic tissue fiber structuresenables precise placement of cells and matrix materials into specificlocations to generate physiologically-relevant heterogeneous meniscusimplants.

In certain embodiments, the mechanical properties of a meniscus implantare controlled by modulating the patterning of collagen, and/or bymodulating one or more characteristics of the matrix materials (e.g.,alginate, collagen) that are used to generate the synthetic tissue fiberstructures. For example, in some embodiments, one or more anchorregions, as described above, are placed about the periphery of animplant to facilitate attachment and/or fixation, e.g., via suturing orthe like. Anchor regions can be generated by the incorporation of higherstrength materials, for example, stiffer synthetic materials, including,but not limited to, polycaprolactone (PCL), poly(lactic-co-glycolicacid) (PLGA), polyurethane (PU) or any combination thereof. Anchorregions in accordance with embodiments of the invention can contain,e.g., double network hydrogels, generated by combining at least twodifferent hydrogel materials including, but not limited to: alginate,Gelatin methacrylol (GelMA), methacryloyl polyethylene glycol (PEGMA),gellan gum, agarose, polyacrylamide, or any combination thereof. Inaddition, high strength fibers may be generated from high concentrationsof biological polymers, including, but not limited to: collagen,chitosan, silk fibroin, or any combination thereof, and these may beincorporated into one or more anchor regions.

Artificial meniscus implants in accordance with embodiments of theinvention can include from 0 to about 12 anchor regions, such as 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or 11 anchor regions. Anchor regions inaccordance with embodiments of the invention can range in size fromabout 5 mm² to about 40 mm², such as about 6, 7, 8, 9 or 10 mm², orabout 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 mm².

Anchor regions in accordance with embodiments of the invention can begenerated by the incorporation of higher strength materials into suturepoints, for example, stiffer synthetic materials such as, e.g.,polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA),polyurethane (PU), or any combination thereof. Anchor regions inaccordance with embodiments of the invention can optionally containdouble network hydrogels, generated by combining at least two differenthydrogel materials, including but not limited to, alginate, Gelatinmethacrylol (GelMA), methacryloyl polyethylene glycol (PEGMA), gellangum, agarose, polyacrylamide, or any combination thereof. In addition,high strength fibers can be generated from high concentrations ofbiological polymers, including, but not limited to, collagen, chitosan,silk fibroin, or any combination thereof. In some embodiments, one ormore of these biological polymers can be incorporated into one or moreanchor regions. In some embodiments, the entire periphery of a layer ofan artificial meniscus implant comprises a reinforced matrix material.In some embodiments, the periphery comprises a plurality of reinforcedanchor regions comprising one or more reinforced matrix materials.

In some embodiments, high strength fibers can be incorporated (e.g.,patterned) into one or more reinforced peripheral regions of anartificial meniscus implant to increase strength along the periphery ofthe implant. In some embodiments, high strength fibers are incorporatedinto the entire periphery of the implant. Within an anchor region and/ora reinforced peripheral region of an artificial meniscus implant, layersof high strength material can be alternated with layers of softermaterial that is optimized for cell survival and ingrowth. Increasedstrength within anchor regions and/or reinforced peripheral region canbe conferred by increasing the concentration of a fiber material, byincreasing the infill density of the printed fibers, by increasing thediameter of the printed fibers, or by any combination thereof. In someembodiments, an anchor region can be colored by incorporating, e.g., anon-toxic dye into the printable anchor material to act as a visualguide during surgery, thereby informing the surgeon of the location ofthe reinforced areas of the artificial meniscus implant that are adaptedfor placement of sutures.

In the human meniscus, the correct orientation and alignment of collagenfibers is crucial to confer appropriate biomechanical properties to thetissue. As discussed previously, spontaneous collagen fiber orientationand subsequent cell alignment can be directed by restricting thecross-linking process to small diameter channels or fibers less thanapproximately 100 μm (Lee et al., 2006) (Onoe et al., 2006). In certainembodiments, the subject meniscus implants comprise a layer wherein oneor more synthetic tissue fiber structures are configured to promotedeposition of collagen fibers that are aligned with a longitudinaldirection of the synthetic tissue fiber. As such, in certainembodiments, a synthetic tissue fiber(s) is deposited in a radial and/ora circumferential orientation, and is configured to promote depositionof collagen fibers that are aligned with the radial and/orcircumferential directional orientation of the synthetic tissuefiber(s). In this way, circumferential and/or radial orientation ofcollagen fibers can be achieved.

In some embodiments, the diameter of a synthetic tissue fiber ismodulated so that collagen fibers are aligned appropriately; e.g. thesurface and periphery of the meniscus contain randomly-oriented (e.g.,disordered) collagen fibers, whereas the inner region(s) containcircumferentially and radially-aligned fibers. An illustration of anon-limiting example of the synthetic tissue fiber orientation in eachof a plurality of layers in a subject meniscus implant is shown in FIG.9 .

Meniscus Injury and Options for Surgical Repair

Damage to the meniscus is very common in the knee joint. Meniscallesions are typically categorized by distinct age groups. Meniscalinjuries in younger human patients (<40 years) are usually caused bytrauma or congenital meniscal diseases, whereas those in older humanpatients (>40 years) tend to be associated with degenerative tears.Meniscal injuries can simply be classified clinically into peripheralmeniscal lesions and avascular meniscal lesions. Numerous surgicaltechniques have been developed to repair meniscal tears in the vascular(red-red) zone with high overall success rates in young patients withstable knees (63-91%). Damage and tearing in the avascular (white-white)zone of the meniscus are often associated with a poor prognosisfollowing repair and consequently several different therapeuticstrategies have been attempted with varied results. The most notableinclude the use of parameniscal synovial tissue, trephination of theperipheral meniscus rim with suture of the meniscus tear, creation ofvascular access channels, and the use of mesenchymal stem cells and/orgrowth factors. None of the above techniques have been generallyadopted, thus the main strategy of orthopedic surgeons is to perform apartial meniscectomy in cases of unrepairable or degenerative meniscalinjuries, even though this treatment strategy does not prevent thedevelopment of knee OA. A partial meniscectomy can result in OA bydecreasing the contact area between the femoral condyle and tibialplatform. Altering the loading characteristics of the articular kneecartilage can lead to progressive degeneration of meniscus and articularcartilage via a vicious cycle of damage, inflammation and further tissuedegeneration.

Artificial Meniscus Implants:

As reviewed above, aspects of the invention include artificial meniscusimplants comprising at least one basal zone, at least one interior zone,and at least one superficial zone, wherein each of said zone comprises alayer comprising at least one synthetic tissue fiber dispensed from abioprinter as a solidified biocompatible matrix optionally comprisingcells, and optionally comprising one or more active agents, as describedherein. In some embodiments, one or more of the matrix material, celltype, cell density, and/or type and/or amount of an active agent canvary can vary across at least one direction of a given layer. Forexample, in some embodiments, a layer of a meniscus implant can have acell density that is lower along a first side, and increases (in alinear or non-linear manner) across the layer towards the opposite side.In certain embodiments, the cell density in a given layer can vary intwo directions. For example, in some embodiments, the cell density in agiven layer can increase (in a linear or non-linear manner) in both anx- and a y-direction across the layer. In certain embodiments, the celldensity can vary from 0 to 100×10⁶ cells per mL, or more.

In some embodiments, at least one layer of the subject artificialmeniscus implant can comprise at least one circumferentially and/orradially oriented synthetic tissue fiber. The circumferential and/orradial fiber(s) can have the same or different diameters, the same ordifferent matrix materials, the same or different cell types, and thesame or different cell densities. In certain embodiments, the diameterof a synthetic tissue fiber can vary from 20 μm to 500 μm.

In certain embodiments, a synthetic tissue fiber is configured topromote deposition of collagen fibers aligned with a longitudinaldirection of the synthetic tissue fiber. In certain embodiments, asynthetic tissue fiber is configured to promote deposition ofrandomly-oriented collagen fibers. As provided in FIG. 10 , collagenfibers in 3D engineered tissues take on an orientation dependent on oneor more features of the scaffold materials used to create the 3D tissue.Similarly, aspects of the subject artificial meniscus implants can bemodulated to control the orientation of the collagen fibers within theimplant material.

In certain embodiments, a subject meniscus implant is constructed, usingsequential deposition of layers, as described above, such that themeniscus implant comprises an inner, central and outer zone, as providedin FIG. 11 . In certain embodiments, the cell type, cell density, and/ormatrix material present in any given zone can be controlled, therebycreating a meniscus implant that resembles the native architecture andbiomechanical characteristics of natural meniscus tissue.

Biocompatible Matrix Materials:

The solidified biocompatible matrix may comprise any of a wide varietyof natural or synthetic polymers that support the viability of livingcells, including, e.g., alginate, laminin, fibrin, hyaluronic acid,poly(ethylene) glycol based gels, gelatin, chitosan, agarose, orcombinations thereof. In preferred embodiments, the solidifiedbiocompatible matrix comprises alginate, or other suitable biocompatiblepolymers that can be instantaneously solidified while dispensing fromthe printhead. In further preferred embodiments, the solidifiedbiocompatible matrix comprises a homogeneous composition of alginatethroughout the radial cross section of each synthetic tissue fiber.

In particularly preferred embodiments, the solidified biocompatiblematrix is physiologically compatible, i.e., conducive to cell growth,differentiation and communication. By “physiological matrix material” ismeant a biological material found in a native mammalian tissue.Non-limiting examples of such physiological matrix materials include:fibronectin, thrombospondin, glycosaminoglycans (GAG) (e.g., hyaluronicacid, chondroitin-6-sulfate, dermatan sulfate, chondroitin-4-sulfate, orkeratin sulfate), deoxyribonucleic acid (DNA), adhesion glycoproteins,and collagen (e.g., collagen I, collagen II, collagen III, collagen IV,collagen V, collagen VI, or collagen XVIII).

Mammalian Cell Types:

Non-limiting examples of mammalian cells types that can be used in thesubject meniscus implants include: fibroblasts, chondrocytes, meniscusfibrochondrocytes, stem cells, bone marrow stromal (stem) cells,embryonic stem cells, mesenchymal stem cells, induced pluripotent stemcells, differentiated stem cells, tissue-derived cells, smooth musclecells, skeletal muscle cells, epithelial cells, endothelial cells,myoblasts, chondroblasts, osteoblasts, osteoclasts, and any combinationsthereof.

Cells can be obtained from donors (allogenic) or from recipients(autologous). Cells can also be from established cell culture lines, orcan be cells that have undergone genetic engineering and/or manipulationto achieve a desired genotype of phenotype. In some embodiments, piecesof tissue can also be used, which may provide a number of different celltypes in the same structure. In one preferred embodiment, the artificialmeniscus implant comprises patient-specific bone marrow-derivedmesenchymal stem cells. In one preferred embodiment, the artificialmeniscus implant comprises primary meniscal chondrocytes. In onepreferred embodiment, the artificial meniscus implant comprisesmicrovascular endothelial cells. In one preferred embodiment, theartificial meniscus implant comprises patient-specific inducedpluripotent stem cell derived chondrocytes.

In some embodiments, cells can be obtained from a suitable donor, eitherhuman or animal, or from the subject into which the cells are to beimplanted. Mammalian species include, but are not limited to, humans,monkeys, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits,rats. In one embodiment, the cells are human cells. In otherembodiments, the cells can be derived from animals such as, dogs, cats,horses, monkeys, or any other mammal.

Without being held to any particular theory, the number of cells seededdoes not limit the final tissue (e.g., meniscus) produced, however,optimal cell density can improve one or more properties of the subjectmeniscus implants.

Cells can be present anywhere within a meniscus implant, e.g., withinthe basal zone, within the interior zone, and/or within the superficialzone. In some embodiments, to mimic native meniscus fibrocartilaginousstructure, different types of cells can be spatially placed in certainzones of the meniscus implant. For example, in some embodiments, one ormore fibroblasts can be placed in a first region and/or in theindividual layers of the meniscus implant. In some embodiments, one ormore chondrocytes can be placed in a first region and/or in theindividual layers of the meniscus implant.

Cells of a particular type, and having a particular density, can beplaced into any desired zone of the subject meniscus implants. In someembodiments, one or more stem cells, e.g., bone marrow stem cells, canbe placed within at least a portion of a subject meniscus implant and/orthe individual layers thereof. In some embodiments, at least a portionof the stem cells can be differentiated to a chondrogenic phenotype. Oneof ordinary skill in the art can readily perform differentiating stemcells into a desired phenotype (e.g., a chondrogenic phenotype) e.g., byexposing the cells to art-recognized cell differentiation factors and/orcommercially-available differentiation media.

Appropriate growth conditions for mammalian cells are well known in theart (Freshney, R. I. (2000) Culture of Animal Cells, a Manual of BasicTechnique. Hoboken N.J., John Wiley & Sons; Lanza et al. Principles ofTissue Engineering, Academic Press; 2nd edition May 15, 2000; and Lanza& Atala, Methods of Tissue Engineering Academic Press; 1st editionOctober 2001). Cell culture media generally include essential nutrientsand, optionally, additional elements such as growth factors, salts,minerals, vitamins, etc., that may be selected according to the celltype(s) being cultured. Particular ingredients may be selected toenhance cell growth, differentiation, secretion of specific proteins,etc. In general, standard growth media include Dulbecco's Modified EagleMedium, low glucose (DMEM), with 110 mg/L pyruvate and glutamine,supplemented with 10-20% fetal bovine serum (FBS) or calf serum and 100U/ml penicillin are appropriate as are various other standard media wellknown to those in the art. Growth conditions will vary dependent on thetype of mammalian cells in use and tissue desired.

Additional sources of human cells include, but are not limited to, bonemarrow-derived mesenchymal stem cells (MSCs) and primary humanmeniscus-derived chondrocytes. MSCs are attractive for regenerativemedicine purposes as they can be isolated from patients and readilyexpanded for use as an autograft tissue replacement. Unlike donorallografts, recipient-derived MSC autografts have zero risk ofinter-person disease transmission or immune-mediated tissue rejection.An additional advantage of MSCs is that the culture protocols todifferentiate MSCs into fibrochondrocyte-like cells are well defined.Chemically defined medium has been demonstrated to induce a chondrogenicphenotype in cultured MSCs as well as promote deposition offibrocartilaginous ECM by MFCs in pellet culture over a 10-week period(Mauck 2006 & Brendon 2007).

The de-differentiation of chondrocytes in 2D cell culture conditions hasencouraged investigation into the effects of more complex physiologicalculture conditions. Oxygen has a fundamental effect on cell behavior andthe cells of the avascular zone of the meniscus are under low oxygenconditions due to the lack of oxygenated blood supply. Several studieshave investigated the effects of hypoxic growth conditions onchondrocyte phenotype; bovine articular chondrocytes grown in hypoxic(5% 02) culture were shown to re-express high amounts of collagen-II atthe protein level compared to the same cells grown in normoxic (21% 02)conditions (Domm et al., 2002). The meniscus is under regularcompressive stress, and it has been postulated that mechanicalstimulation is necessary to trigger appropriate chondrocyte phenotype.Ultrasonic stimulation at a frequency of 1 MHz was demonstrated toincrease the deposition of ECM by chondrocytes in 2D and 3D cultures,but the effect was transient only lasting for 28 days (Hsu et al.,2006). Upton et al., isolated cells from the inner and outer zones ofthe meniscus and grew them in monolayers on flexible membranes. Whenexposed to a biaxial strain of 5% both populations of cells were shownto increase NO and total protein expression (Upton et al., 2006). Foroptimal meniscus biomechanical performance, hypoxic culture andmechanical strain can be utilized to maximize phenotypic differentiationof MSC-derived chondrocytes or primary meniscal cells in 3D cultures.

Active Agents:

In some aspects, a meniscus implant in accordance with embodiments ofthe invention can comprise at least one active agent. Non-limitingexamples of such active agents include TGF-β1, TGF-β2, TGF-β3, BMP-2,BMP-4, BMP-6, BMP-12, BMP-13, basic fibroblast growth factor, fibroblastgrowth factor-1, fibroblast growth factor-2, platelet-derived growthfactor-AA, platelet-derived growth factor-BB, platelet rich plasma,IGF-I, IGF-II, GDF-5, GDF-6, GDF-8, GDF-10, vascular endothelialcell-derived growth factor, pleiotrophin, endothelin, nicotinamide,glucagon like peptide-I, glucagon like peptide-II, parathyroid hormone,tenascin-C, tropoelastin, thrombin-derived peptides, laminin, biologicalpeptides containing cell-binding domains and biological peptidescontaining heparin-binding domains, therapeutic agents, and anycombinations thereof.

The term “therapeutic agents” as used herein refers to any chemicalmoiety that is a biologically, physiologically, or pharmacologicallyactive substance that acts locally or systemically in a subject.Non-limiting examples of therapeutic agents, also referred to as“drugs”, are described in well-known literature references such as theMerck Index, the Physician's Desk Reference, and The PharmacologicalBasis of Therapeutics, and they include, without limitation,medicaments; vitamins; mineral supplements; substances used for thetreatment, prevention, diagnosis, cure or mitigation of a disease orillness; substances which affect the structure or function of the body;or pro-drugs, which become biologically active or more active after theyhave been placed in a physiological environment. In some embodiments,one or more therapeutic agents can be used, which are capable of beingreleased from a meniscus implant described herein into adjacent tissuesor fluids upon implantation to a subject. Examples of therapeutic agentsinclude, but are not limited to, antibiotics, anesthetics, anytherapeutic agents that promote meniscus regeneration or tissue healing,or that reduce pain, infection, or inflammation, or any combinationthereof.

Additional active agents can include, but are not limited to, proteins,peptides, nucleic acid analogues, nucleotides, oligonucleotides, nucleicacids (DNA, RNA, siRNA), peptide nucleic acids, aptamers, antibodies orfragments or portions thereof, antigens or epitopes, hormones, hormoneantagonists, growth factors or recombinant growth factors and fragmentsand variants thereof, cytokines, enzymes, antibiotics or antimicrobialcompounds, anti-inflammation agent, antifungals, antivirals, toxins,prodrugs, small molecules, drugs (e.g., drugs, dyes, amino acids,vitamins, antioxidants) or any combination thereof.

Non-limiting examples of antibiotics that are suitable for inclusion ina meniscus implant of the present invention include: aminoglycosides(e.g., neomycin), ansamycins, carbacephem, carbapenems, cephalosporins(e.g., cefazolin, cefaclor, cefditoren, cefditoren, ceftobiprole),glycopeptides (e.g., vancomycin), macrolides (e.g., erythromycin,azithromycin), monobactams, penicillins (e.g., amoxicillin, ampicillin,cloxacillin, dicloxacillin, flucloxacillin), polypeptides (e.g.,bacitracin, polymyxin B), quinolones (e.g., ciprofloxacin, enoxacin,gatifloxacin, ofloxacin, etc.), sulfonamides (e.g., sulfasalazine,trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole)),tetracyclines (e.g., doxycyline, minocycline, tetracycline, etc.),chloramphenicol, lincomycin, clindamycin, ethambutol, mupirocin,metronidazole, pyrazinamide, thiamphenicol, rifampicin, thiamphenicl,dapsone, clofazimine, quinupristin, metronidazole, linezolid, isoniazid,fosfomycin, fusidic acid, or any combination thereof.

Non-limiting examples of antibodies include: abciximab, adalimumab,alemtuzumab, basiliximab, bevacizumab, cetuximab, certolizumab pegol,daclizumab, eculizumab, efalizumab, gemtuzumab, ibritumomab tiuxetan,infliximab, muromonab-CD3, natalizumab, ofatumumab omalizumab,palivizumab, panitumumab, ranibizumab, rituximab, tositumomab,trastuzumab, altumomab pentetate, arcitumomab, atlizumab, bectumomab,belimumab, besilesomab, biciromab, canakinumab, capromab pendetide,catumaxomab, denosumab, edrecolomab, efungumab, ertumaxomab,etaracizumab, fanolesomab, fontolizumab, gemtuzumab ozogamicin,golimumab, igovomab, imciromab, labetuzumab, mepolizumab, motavizumab,nimotuzumab, nofetumomab merpentan, oregovomab, pemtumomab, pertuzumab,rovelizumab, ruplizumab, sulesomab, tacatuzumab tetraxetan, tefibazumab,tocilizumab, ustekinumab, visilizumab, votumumab, zalutumumab,zanolimumab, or any combination thereof.

Non-limiting examples of enzymes suitable for use in a meniscus implantas described herein include: peroxidase, lipase, amylose,organophosphate dehydrogenase, ligases, restriction endonucleases,ribonucleases, DNA polymerases, glucose oxidase, and laccase.

Additional non-limiting examples of active agents that are suitable foruse with the subject meniscus implants include: cell growth media, suchas Dulbecco's Modified Eagle Medium, fetal bovine serum, non-essentialamino acids and antibiotics; growth and morphogenic factors such asfibroblast growth factor, transforming growth factors, vascularendothelial growth factor, epidermal growth factor, platelet derivedgrowth factor, insulin-like growth factors), bone morphogenetic growthfactors, bone morphogenetic-like proteins, transforming growth factors,nerve growth factors, and related proteins (growth factors are known inthe art, see, e.g., Rosen & Thies, CELLULAR & MOLECULAR BASIS BONEFORMATION & REPAIR (R.G. Landes Co., Austin, Tex., 1995);anti-angiogenic proteins such as endostatin, and other naturally derivedor genetically engineered proteins; polysaccharides, glycoproteins, orlipoproteins; anti-infectives such as antibiotics and antiviral agents,chemotherapeutic agents (i.e., anticancer agents), anti-rejectionagents, analgesics and analgesic combinations, anti-inflammatory agents,steroids, or any combination thereof

Methods for Repairing a Meniscal Defect:

Aspects of the invention include methods for repairing and/or replacingat least a portion of a meniscus in a subject. Any of the meniscusimplants described herein can be implanted into a subject in needthereof in order to accomplish meniscus repair or regeneration.Accordingly, methods of repairing a meniscal defect or promotingmeniscal regeneration in a subject are also provided herein. In oneembodiment, a method comprises implanting a meniscus implant asdescribed herein into a defect site in need of meniscus repair orregeneration.

The term “subject” includes, but is not limited to, humans, nonhumanprimates such as chimpanzees and other apes and monkey species; farmanimals such as cattle, sheep, pigs, goats and horses; domestic mammalssuch as dogs and cats; laboratory animals including rodents such asmice, rats and guinea pigs, and the like. The term does not denote aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are included. In one embodiment, thesubject is a mammal. In one embodiment, the subject is a human subject.

In some embodiments, a method can comprise securing a meniscus implant,or an anchor region thereof, at a defect site, and/or securing one ormore anchor regions of a meniscus implant to at least one anatomicalstructure within a subject. In some embodiments, a method can furthercomprise removing at least a portion of a defective meniscus from thesubject.

In some embodiments, a method can further comprise systemically and/orlocally (e.g., to the meniscus implant site) administering to thesubject at least one active agent described herein.

All patents and patent publications referred to herein are herebyincorporated by reference in their entirety.

Certain modifications and improvements will occur to those skilled inthe art upon a reading of the foregoing description. It should beunderstood that not all such modifications and improvements have beenincluded herein for the sake of conciseness and readability, but areproperly within the scope of the following claims.

What is claimed:
 1. A synthetic tissue structure comprising a pluralityof layers deposited by a bioprinter, each layer comprising one or moresynthetic tissue fibers comprising a solidified biocompatible matrix,wherein the type of matrix material varies in at least one directionwithin one or more synthetic tissue fibers in at least one layer, andwherein said tissue structure further comprises one or more reinforcedperipheral regions.
 2. The synthetic tissue structure according to claim1, wherein at least one of said layers comprises a single continuoussynthetic tissue fiber dispensed from the bioprinter.
 3. The synthetictissue structure according to claim 2, wherein the single continuoussynthetic tissue fiber dispensed from the bioprinter has a variablecomposition.
 4. The synthetic tissue structure according to claim 1,wherein each of said layers comprises a matrix material that varies intype in at least one direction.
 5. The synthetic tissue structureaccording to claim 1, wherein the periphery of said synthetic tissuestructure comprises a plurality of reinforced anchor regions comprisingone or more reinforced matrix materials.
 6. The synthetic tissuestructure of claim 5, wherein the one or more anchor regions arecomprised of polycaprolactone (PCL), poly(lactic-co-glycolicacid)(PLGA), polyurethane (PU), or any combination thereof, or one ormore double network hydrogels.
 7. The synthetic tissue structure ofclaim 5, wherein the one or more anchor regions further comprise anon-toxic dye.
 8. The synthetic tissue structure according to claim 1,wherein the entire periphery of a layer of said synthetic tissuestructure comprises a reinforced matrix material.
 9. The synthetictissue structure of claim 1, wherein the matrix material comprisesalginate, laminin, fibrin, hyaluronic acid, poly(ethylene) glycol basedgel(s), gelatin, chitosan, agarose, or a combination thereof.
 10. Thesynthetic tissue structure of claim 9, wherein the matrix materialcomprises alginate.
 11. The synthetic tissue structure of claim 1,wherein the solidified biocompatible matrix is physiologicallycompatible.
 12. The synthetic tissue structure of claim 11, wherein thesolidified biocompatible matrix comprises one or more of collagen,fibronectin, thrombospondin, glycosaminoglycans (GAG), deoxyribonucleicacid (DNA), adhesion glycoproteins, elastin, and combinations thereof.13. The synthetic tissue structure of claim 12, wherein the collagen iscollagen I, collagen II, collagen III, collagen IV, collagen V, collagenVI, or collagen XVIII.
 14. The synthetic tissue structure of claim 12,wherein the GAG is hyaluronic acid, chondroitin-6-sulfate, dermatansulfate, chondroitin-4-sulfate, or keratin sulfate.
 15. The synthetictissue structure of claim 1, further comprising cells.
 16. The synthetictissue structure of claim 15, wherein at least one of said layerscomprises a cell type and/or a cell density that varies in at least onedirection within the one or more synthetic tissue fibers.
 17. Thesynthetic tissue structure of claim 15, wherein the cells are mammaliancells; and wherein the mammalian cells are selected from the groupconsisting of fibroblasts, chondrocytes, fibrochondrocytes, primaryhuman meniscus-derived chondrocytes, stem cells, bone marrow cells,embryonic stem cells, mesenchymal stem cells, bone marrow-derivedmesenchymal stem cells, induced pluripotent stem cells, differentiatedstem cells, tissue-derived cells, microvascular endothelial cells, andcombinations thereof.
 18. The synthetic tissue structure of claim 1,further comprising one or more active agents.
 19. The synthetic tissuestructure of claim 18, wherein at least one of said layers comprises oneor more active agents that vary in type and/or amount in at least onedirection within the one or more synthetic tissue fibers.
 20. Thesynthetic tissue structure of claim 1, wherein the one or morereinforced peripheral regions comprise one or more layers of higherstrength material(s) deposited in alternation with one or more layers ofsofter matrix materials, wherein the softer matrix materials comprisematerials conducive to cell survival and ingrowth.
 21. The synthetictissue structure of claim 20, wherein the one or more layers of higherstrength materials are comprised of polycaprolactone (PCL),poly(lactic-co-glycolic acid)(PLGA), polyurethane (PU), or anycombination thereof, or one or more double network hydrogels generatedby combining at least two different hydrogel materials.
 22. Thesynthetic tissue structure of claim 21, wherein the at least twodifferent hydrogel materials include alginate, Gelatin methacrylol(GelMA), methacryloyl polyethylene glycol (PEGMA), gellan gum, agarose,polyacrylamide, or any combination thereof.
 23. The synthetic tissuestructure of claim 1, wherein the one or more reinforced peripheralregions comprises a non-toxic dye.